The proposed project will address the general question of the factors involved in protein subunit interactions and consequential biological activities. The pyridine nucleotide-linked D-lactate dehydrogenases present a valuable system for considering this question because of their clear homology with the well-characterized L-lactate dehydrogenases. However, in addition to their clear difference in lactate stereospecificity, the two types of enzymes exhibit different subunit association phenomena: the D-lactate dehydrogenases exist as dimers, whereas the L-stereospecific enzymes are ordinarily found as tetramers. Studies will center around the best characterized of the D-lactate dehydrogenases, that from the muscle of the horseshoe crab, Limulus polyphemus. Physico-chemical studies of the forces involved in conferring appropriate conformation(s) and subsequent subunit associations will be studied. Analyses of the subunit and multisubunit structures will employ fluorometric and circular dichroic spectra, amino acid composition and sequence determinations, and immunological cross reactivity. An important aspect of these studies will be the in vitro hybridization of isolated D- and L-lactate specific subunits, and the structural and enzymatic analyses of the resulting molecules. Special attention will be paid to the comparison of the subunit interactions in the dimeric D-lactate dehydrogenases and the tetrameric L-lactate dehydrogenases. It is anticipated that the careful study of the D- and L-lactate dehydrogenases, and the reversible dissociation-reassociation phenomena in vitro will help to elucidate the factors involved in yielding biologically active multisubunit proteins. To facilitate these studies more effective procedures for purifying large amounts of protein will be developed. One additional aspect of the proposed research will be the search for the characterization of alternative D-lactate dehydrogenases for the above comparative biochemistry. This study may aid in the analysis of subunit interactions for other globular proteins.